West Nile virus has become an increasing public health concern in the United States since its appearance in New York City in 1999. In order to gain a better understanding of the interaction of this virus with the infected host cell, we have performed gene microarray studies on WNV infected cells. The results of these studies suggested that the src-family kinase c-Yes may play a role in WNV infection. In agreement with this hypothesis, addition of the src-family kinase inhibitors PP2 and SU6656 to infected cells resulted in a 10-20 fold decrease in the amount of virus recovered in the culture supernatant 20 h after drug addition, and a rapid decrease of cell associated infectious virus soon (4 h) after drug addition. Interestingly, drug treatment does not result in a corresponding decrease in the amount of viral RNA within the infected cell, suggesting that c-Yes, or other src kinases, do not act on viral RNA replication, but at a later stage in the viral life cycle. Specific inhibition of c-Yes in an siRNA mediated "knock-down" experiment also resulted in a decrease in recovered virus, indicating that c-Yes is indeed involved in WNV replication, although a role for other members of the src family cannot be excluded. The aim of this application is to extend these studies to determine what members of the src kinases, other than c-Yes, are also involved in WNV replication, where in the viral life cycle the effect of c-Yes is exerted, and what substrates, viral or cellular, are phosphorylated by the kinase during infection. [unreadable] [unreadable]